Elite controllers are HIV-1 infected persons with undetectable viral loads in the absence of HAART, and the identification of immune defense mechanisms in these patients may reveal critical new information for increasing immune defense against HIV-1 in a broader HIV-1 patient population. Previous studies in elite controllers have mostly focused on classical T- and B-cell mediated immune responses against HIV-1. However, it has been shown that such adaptive immune activity may not be a sufficient, and sometimes not even a necessary component of effective immune protection against HIV-1 in these patients. In preliminary studies, we found that CD4 T cells from elite controllers are substantially less susceptible to HIV-1 replication. This partial resistance of CD4 T cells from elite controllers was mediated by a strong upregulation of the cyclin- dependent kinase inhibitor p21 (waf-1/cip-1), which was able to independently suppress HIV-1 reverse transcription and mRNA transcription. To our knowledge, these data represent the first description of a molecular HIV-1 restriction factor that actively inhibits HIV-1 replication in vivo in elite controllers and an therefore serve as a direct molecular target for enhancing host resistance to HIV-1. Here, we propose to extend our investigations of p21-mediated HIV-1 restriction by strategically focusing on three specific aims: To analyze molecular pathways responsible for the protective high-level expression of p21 in CD4 T cells from elite controllers, we will investigate how miRNAs regulate p21 gene transcription in CD4 T cells, using longitudinal samples collected from a unique cohort of HIV-1 elite controllers for whom PBMC samples are available from the time of acute infection (specific aim 1). In specific aim 2, we propose to mechanistically investigate how p21 affects HIV-1 reverse transcription, one of the earliest and most important HIV-1 replication steps. We hypothesize that p21 can indirectly inhibit HIV-1 reverse transcriptase by blocking host proteins from the cyclin-dependent kinase family that activate reverse transcriptase. If these studies are successful, we will identify a new aspect of HIV-1 host-pathogen interactions that can intercept an early HIV-1 replication step and may therefore be particularly attractive for clinical approaches to increase host resistance to HIV-1. Finally, we will mechanistically investigate how p21 may contribute to mechanisms of HIV-1 latency by affecting host factors involved in transcriptional elongation of HIV-1 mRNA transcripts; these investigations may provide important new information for inducing a long-term drug-free remission of HIV-1 infection by preventing viral outgrowth from latently infected cells (specific aim 3). Overall, these studies represent a highly interdisciplinary investigation integrating immunologic, virologic and biochemical approaches for the mechanistic investigation of a novel HIV-1 restriction factor that is effective at limiting the susceptibility to HIV-1 infection in vivo in elite controllers; if sucessful, these studies will reveal important new information for HIV-1 pathogenesis, immune defense and clinical strategies for prevention and treatment of HIV-1 infection.